Licheninase

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2016 – 23 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted 28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate issued by Health Care Inspectorate of the Ministry of Health, The Netherlands.

Test material

Test material information

Constituent 1

Reference substance name:

Licheninase

EC Number:

819-930-0

Cas Number:

37288-51-0

Molecular formula:

Not applicable, please see remarks

IUPAC Name:

Licheninase

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPB41588
- Expiration date of the lot/batch: 05-07-2026
- Storage condition of test material: < -10⁰C

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ reconstructed skin membranes

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

other: PBS

Details on test system:

SKIN DISC PREPARATION
- Procedure used: EpiDerm™ reconstructed skin membranes. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2).
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC and 5% CO2
- Temperature of post-treatment incubation (if applicable): Same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: each skin model was removed from the well, rinsed with PBS to remove the study substance (and mesh), blotted dry and transferred to a 6-well plate containing medium.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Yes, but not specified.
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
The in vitro skin irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
Mean tissue viability (% of negative control): ≤ 50 %, classification and labelling required, irritant or corrosive.
Mean tissue viability > 50 %, Non-irritant (No Category).

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate tissues each for test substance, negative control Phosphate Buffered Saline and positive control 5% Sodium Dodecyl Sulphate.
For test enzyme licheninase, one replicate was excluded due to a notably insufficient extraction of the MTT.

Test system

Type of coverage:

open

Preparation of test site:

other: Not necessary since it is a three-dimensional human skin model.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

Duration of treatment / exposure:

60 minutes

Details on study design:

TEST SITE
- Area of exposure: 0.64 cm2
- % coverage: 100%
- Type of wrap if used: Nylon mesh

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With PBS
- Time after start of exposure: Exposure time 60 min, post-exposure time approx. 42 h.

SCORING SYSTEM:
- Method of calculation: OD measurement with a spectrophotometer

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No reduction was observed.
- Colour interference with MTT: None observed.

The experiment was considered valid if:
- the OD of the viable negative control was ≥ 0.8 and ≤ 2.8.
- skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control.

The test group was considered valid if the standard deviation (SD) calculated from individual tissue viability percentages of the three replicates was ≤ 18%.
- Range of historical values if different from the ones specified in the test guideline: Although the data are not within the historical range (on the edge), they are within the criteria. Since the acceptance criteria were met, the experiment was considered valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of the skin membranes was 98 ± 9% compared to the negative control group. Based on the results obtained in the present study, the test substance licheninase, batch PPB41588, was classified as non-irritant.

Executive summary:

This study was performed to determine the in vitro skin irritation potential of licheninase, BatchPPB41588, using EpiDerm™ reconstructed skin membranes.

The skin membranes were topically exposed to the test substance for 60 min. Viability of the epidermal cells was assessed using the MTT test at ca. 42 h post-exposure. Negative control (PBS) and positive control (5% SDS) were run in parallel. The general principle for the detection of viability via the MTT test is the conversion of the yellow tetrazolium salt (MTT) to the blue/purple coloured product formazan by mitochondrial enzymes. The formation of formazan was measured using a spectrophotometer.

The acceptance criteria of the negative and positive control were met and therefore the study was considered valid.

The mean viability of the skin membranes was 98 ± 9% compared to the negative control group. One replicate was excluded due to a notably insufficient extraction of the MTT.

Based on the results obtained in the present study, the test substance licheninase, batch PPB41588, was classified as non-irritant (UN GHS No Category).